PROJECT SUMMARY Histoplasma capsulatum is a fungal pathogen that infects both immunocompromised and immunocompetent individuals. The innate immune system alone is ineffective in controlling Histoplasma yeasts because Histoplasma invades and thrives within host phagocytic cells. Histoplasma?s parasitism of macrophages requires acquisition and metabolism of suitable nutritional resources from the host cell, yet the host molecules that serve as nutrients for are largely unknown. Preliminary investigations have shown intracellular yeasts are gluconeogenic and likely consume amino acids, particularly compounds that are related to glutamate. These findings provide a critical foothold for identifying the host molecules consumed by Histoplasma yeasts. This proposal takes a multidisciplinary approach, integrating evidence from molecular biology, genetics, and metabolomics to determine the host molecules available to Histoplasma yeasts within the phagosome and how they are metabolized to meet the carbon and energy needs within host cells. The intracellular growth requirement for glutamate dehydrogenase activity narrows the potential host molecules for metabolic carbon to glutamate-related amino acids or glutamate-generating host molecules. Transcriptional and metabolite profiling will be combined with functional tests to define the metabolic pathways necessary for Histoplasma proliferation within host macrophages. Isotopic labeling of host metabolites will be used to follow their import and subsequent incorporation into Histoplasma metabolism intermediates over time to demonstrate the flux of carbon from macrophage to intracellular yeasts and identify host substrate entry points into yeast central carbon metabolism. These findings will be genetically tested by characterization of Histoplasma?s amino acid and peptide transporters and RNAi-based interference with candidate host metabolite import into intracellular Histoplasma yeast cells. Together these data will provide multiple lines of evidence to define how Histoplasma yeasts exploit the phagosome as a replication-permissive intracellular niche during host infection. This proposal is submitted in response to Funding Opportunity Announcement (FOA) PA-19-083 ?Novel approaches to understand, prevent, treat, and diagnose coccidioidomycosis (Valley Fever) and other select endemic fungal infections.? The proposal answers the announcement?s goal to address the pathogenesis of endemic fungi with the ultimate goal of advancing the field towards solutions for treatment of endemic mycoses. Of the dimorphic endemic fungal pathogens, Histoplasma is the best molecularly characterized fungus and it has the most advanced molecular genetics to facilitate functional testing for mechanistic studies. The results of our studies will reveal new disease treatment avenues by highlighting transport and metabolic reactions essential for the narrow metabolism imposed on Histoplasma by its residence within macrophage phagosomes. Our genetic tests have shown that impairment of these critical metabolic reactions reduces Histoplasma proliferation in the mammalian lung, demonstrating the therapeutic potential.